The three-dimensional structure of the wild type and mutants of homeodomains in the NK-2 class, specifically the vnd/NK-2 homeodomain, and the full length CSX/NKX-2.5, and NKX-3.1 proteins, both in the free state and bound to DNA have been investigated. In addition, transgenic studies involving three conserved region deletion mutants (i.e., tinman, the NK-2 box that encodes the NK-2 specific domain, and the acidic box that encodes the acidic domain) and a single amino acid residue replacement were carried out. The single amino acid residue replacement involved replacing tyrosine with methionine in position 54 of the homeodomain (i.e., Y54M). The tyrosine is the single most important variable amino acid residue responsible for the recognition of a DNA sequence that contains 5' - CAAGTG - 3' as its core. With regard to the transgenic studies, the ectopic expression patters of the wild type and the four mutant vnd/NK-2 genes were analyzed together with the expression of two down-stream target genes, ind and msh, that are down-regulated by vnd/NK-2. Of the four ectopically expressed mutant genes examined, only the Y54M alteration acted as a knockout, where ability to down-regulate ind and msh apparently is suppressed completely. The acidic domain deletion mutant showed limited down-regulation capability. By contrast, the tinman and the NK-2 box deletion mutants both behaved fully as functional vnd/NK-2 genes in their ability to repress ind and msh. The NMR determined tertiary structures of the Y54M vnd/NK-2 homeodomain in vitro both free and bound to DNA were compared with the wild type analog. The only structural difference observed for the mutant homeodomain was in the complex with DNA and involved a closer interaction of the methionine with A2 rather that C3 of the DNA. This subtle change in the homeodomain-DNA complex resulted in modifications of the binding affinities of the Y54M vnd/NK-2 homeodomain to DNA. These changes resulting from a single amino acid residue replacement constitute the molecular basis for the phenotypic alterations observed upon ectopic expression of the Y54M vnd/NK-2 gene during embryogenesis. An important relationship between the regulatory role of the NK-2 class of homeobox genes in development and highly explicity homeodomain-DNA interactions is thereby established. These studies also demonstrate the relation between sequential and structural modularity with functional modularity, where in the above example, only the homeodomain appears to be necessary to down-regulate ind and msh. Studies on mutant CSX/NKX-2.5 homeodomains associated with atrial septum defect show that the tertiary structure of the Y54C mutant protein is not altered but that the interaction with the functional DNA binding sites is modified. The Y54C CSX/NKX-2.5 protein is totally non-functional. This modification of the interaction of the protein with the DNA likely constitutes the molecular basis of the disease. Recent structural studies suggest that that only part of the CSX/NKX-2.5 protein that is structured in the absence of binding to DNA or other protein factors is the homeodomain itself.